Cytochrome P450 monooxygenases consisting of thermophilic bacteria

ABSTRACT

The invention relates to novel cytochrome P450 monooxygenases from thermophilic bacteria, in particular the genus  Thermus  sp., to nucleotide sequences encoding them, to the recombinant production of these monooxygenases and to their use for the microbiological oxidation of organic compounds.

The invention relates to novel cytochrome P450 monooxygenases fromthermophilic bacteria, in particular from the genus Thermus sp., tonucleotide sequences encoding them, to the recombinant preparation ofthese monooxygenases, and to their use for the microbiological oxidationof organic compounds.

Cytochrome P450 monooxygenases have the ability of catalyzingoxygenation reactions which are of industrial interest and havetherefore been researched intensively for some time. Thus, for example,the cytochrome P450 monooxygenase BM-3 has been isolated from Bacillusmegaterium and characterized and can now be obtained by the recombinantroute (cf., for example, DE-A-199 35 115).

This cytochrome P450-monooxygenase usually catalyzes the subterminalhydroxylation of long-chain, saturated acids and of the correspondingamides and alcohols thereof or the epoxidation of unsaturated long-chainfatty acids or saturated fatty acids with medium chain length. Theoptimal chain length of saturated fatty acids is 14 to 16 carbon atoms.

The structure of the heme domain of P450 BM-3 was determined by X-raystructural analysis. The substrate binding site is in the form of a longtunnel-like opening which reaches from the surface of the molecule tothe heme molecule and is delimited virtually exclusively by hydrophobicamino acid residues. The only charged residues at the surface of theheme domain are the residues Arg47 and Tyr51. It is assumed that thelatter participate in the binding of the carboxylate group of thesubstrate by forming a hydrogen bond. In the meantime, the substratespectrum of this enzyme has been widened successfully by the targetedintroduction of point mutations. Thus, the oxidation of both shorter-and longer-chain carboxylic acids, alkanes, alkenes, cycloalkanes,cycloalkenes and a wide range of aromatics by this enzyme is nowpossible (cf. DE-A-199 35 115, 199 55 605, 100 11 723 and 100 14 085).

To improve the industrial applicability of this class of enzymesfurther, it would therefore be desirable to find novel cytochrome P450monooxygenases which are better adapted to industrial productionconditions, such as, for example, enzymes with increased thermalstability.

The object of the present invention was therefore to provide cytochromeP450 monooxygenases which are adapted better to industrial productionconditions.

The above object was achieved by providing a cytochrome P450monooxygenase which comprises an amino acid sequence encompassing asubsequence from the amino acid residue Pro328 to Glu345 in accordancewith SEQ ID NO:2 and preferably also a subsequence from the amino acidresidue Val216 to Ala227 in accordance with SEQ ID NO:2.

Cytochrome P450 monooxygenases which are preferred in accordance withthe invention have an amino acid sequence encompassing at least onefurther subsequence which is selected from among a subsequence of atleast 10 successive amino acids from the sequence regions predeterminedby the amino acid residues Met1 to Phe327 and Gly346 to Ala389 inaccordance with SEQ ID NO:2.

An especially preferred cytochrome P450 monooxygenase has an amino acidsequence which corresponds essentially to SEQ ID NO: 2.

Cytochrome P450 monooxygenases according to the invention can beisolated in particular from thermophilic bacteria, preferably of thegenus Thermus sp., such as, for example, the species Thermusthermophilus, strain HB27 (deposited at the DSM under Number DSM7039).In accordance with the invention, “Thermophilic” bacteria meet thetemperature tolerance criteria of H. G. Schlegel, AllgemeineMikrobiologie [General Microbiology], Thieme Verlag Stuttgart, 5thEdition, page 173, for thermophiles and extreme thermophiles (i.e.growth optimum at over 40° C.).

The monooxygenases according to the invention are preferablycharacterized by an increased thermostability. This takes the form of alower loss of activity at elevated temperature compared to the Bacillusmegaterium P450 BM-3 (for example in a range of from 30 to 60° C., pH7.5, 25 mM Tris/HCl).

In accordance with a preferred embodiment, a cytochrome P450monooxygenase is provided in accordance with the invention from thethermophilic bacterium T. thermophilus. The protein has a molecularweight of approximately 44 kDa (determined by SDS gel electrophoresis),is soluble, and has an absorption spectrum in the reduced state,oxidized state, and as carbonyl adduct which is analogous to that of theother P450 enzyme. The following identities were determined fromsequence alignments of this enzyme according to the invention from T.thermophylus and other known P450 enzymes: P450 BM3, 32% identity;CYP119, 29% identity; P450eryF, 31% identity. The enzyme according tothe invention has extraordinary thermostability, which is demonstratedby a melting temperature of approximately 85° C., which value is 30° C.above that of P450cam.

The subject matter of the invention are furthermore oligonucleotideswhich hybridize with a nucleic acid sequence encoding a cytochrome P450monooxygenase according to the invention.

In particular, the subject matter of the invention are also thoseoligonucleotides which encompass a nucleic acid sequence which isessentially complementary to a nucleotide sequence region in accordancewith SEQ ID NO:1 which encompasses at least 30 to 45 successivenucleotide residues.

A further subject matter of the invention relates to polynucleotideswhich hybridize with an oligonucleotide as defined above and whichencode a cytochrome P450 monooxygenase, in particular a cytochrome P450monooxygenase from other microorganisms, such as, for example, those ofthe genus Thermus sp.

The subject matter of the present invention are, in particular, alsopolynucleotides which encode a cytochrome P450 monooxygenase as definedabove, and polynucleotides which are complementary thereto.

Preferred polynucleotides are those which have essentially a nucleicacid sequence in accordance with SEQ ID NO: 1, and the nucleic acidsequences which are complementary thereto and derived therefrom.

A further subject matter of the invention relates to expressioncassettes for the recombinant production of monooxygenases according tothe invention, comprising at least one regulatory nucleic acid sequencelinked operably to at least one of the polynucleotides stated above.

Further subject matters of the invention relate to recombinant vectorswhich carry at least one polynucleotide or at least one expressioncassette as defined above; and to microorganisms comprising at least onesuch recombinant vector; and to processes for the preparation ofcytochrome P450 monooxygenases according to the invention, in which amicroorganism which produces cytochrome P450 monooxygenase is culturedand the monooxygenase is isolated from the culture.

The enzymes according to the invention and mutants which can be derivedtherefrom are useful as biocatalysts for various biochemical oxygenationreactions of organic compounds of industrial importance. Analogously,the recombinant microorganisms according to the invention can also beemployed for carrying out such oxygenation reactions.

A further subject matter of the invention therefore relates to a processfor the microbiological oxidation of an organic compound, wherein thiscompound is reacted with at least one cytochrome P450 monooxygenaseaccording to the invention.

This process is preferably carried out in such a way that

-   a1) a recombinant microorganism as defined above is cultured in a    culture medium in the presence of the exogenous (externally    supplied) organic compound, or the organic compound which has been    formed as intermediate, which compound is a substrate for    monooxygenase, preferably in the presence of oxygen and if    appropriate an electron donor; or-   a2) a substrate-containing reaction medium is incubated with a    cytochrome P450 monooxygenase according to the invention, preferably    in the presence of oxygen and an electron donor; and-   b) the oxidation product formed or a subsequent product thereof is    isolated from the medium.

The exogenous substrate, or the substrate which has been formed asintermediate, can be selected from among:

-   a) optionally substituted N-, O- or S-heterocyclic mono-, di- or    polynuclear aromatic compounds;-   b) optionally substituted mono- or polynuclear aromatics;-   c) straight-chain or branched alkanes and alkenes;-   d) optionally substituted cycloalkanes and cycloalkenes; and-   e) aliphatic, preferably terminally saturated, carboxylic acids.

In a first preferred variant of the process according to the invention,the oxidation is carried out by culturing the microorganisms in thepresence of oxygen at a culture temperature of at least approximately20° C. and a pH of approximately 6 to 9.

In a second preferred variant of the process according to the invention,at least one compound selected from among the above-defined groups a) toe) is added as exogenous substrate to a medium and the oxidation iscarried out by enzymatic conversion of the substrate-containing mediumin the presence of oxygen at a temperature of at least approximately 20°C. and a pH of approximately 6 to 9, wherein the substrate-containingmedium additionally contains an approximately 10- to 100-fold molarexcess of reduction equivalents (electron donor) based on the substrate.

The above processes can preferably be carried out in bioreactors. Thesubject matter of the invention are therefore such bioreactors,comprising at least one monooxygenase according to the invention or atleast one recombinant microorganism, if appropriate in each case inimmobilized form.

Finally, the invention relates to the use of a cytochrome P450monooxygenase, of a vector or a microorganism according to the presentinvention for the microbiological oxidation of the abovementionedclasses of organic compounds.

The invention is now illustrated in greater detail with reference to theappended figures. In these figures,

FIG. 1 shows a P450 Thermus thermophilus with the heme domain ofBacillus megaterium P450 BM3. The heme binding site is shown doublyunderlined (Cys400 in P450 BM3 is the cystein residue which coordinateswith the iron atom of the prosthetic group). The region which is incontact with the ω-end of the fatty acid chain is singly underlined. Theextent of their agreement is designated by different symbols(“*”=identical residues; “:” and “.”=similar residues).

FIG. 2 shows the result of a comparison test for determining thethermostability of P450 BM3 and Thermus sp. P450. The thermostabilitywas determined spectrometrically in a wavelength range between 400 and500 nm over the heme group content.

Also encompassed in accordance with the invention are “functionalequivalents” of the new P450 monooxygenases which have been disclosedspecifically.

“Functional equivalents” or analogs of the monooxygenases which havebeen disclosed specifically are, for the purposes of the presentinvention, enzymes which differ from the above and which continue toshow the desired substrate specificity within the scope of at least oneof the above-designated oxidation reactions a) to e) and/or show anincreased thermostability in comparison with P450 BM3, for example attemperatures in the range of approximately 30 to 60° C. and, ifappropriate, higher temperatures after treatment for 30 minutes in 25 mMTris/HCl.

“Functional equivalents” are understood as meaning in accordance withthe invention in particular mutants which exhibit an amino acid otherthan the amino acid mentioned specifically in at least one of theabovementioned sequence positions but which still catalyze one of theabovementioned oxidation reactions. “Functional equivalents” thus alsoencompass the mutants which are obtainable by one or more, such as forexample 1 to 30 or 1 to 20 or 1 to 10, amino acid additions,substitutions, deletions and/or inversions, it being possible for theabovementioned modifications to occur in any sequence position as longas they lead to a mutant with the spectrum of properties according tothe invention. Functional equivalence exists in particular also when thereactivity patterns between mutant and unmodified enzyme agree in termsof quality, i.e. when identical substrates are converted at differentrates.

“Functional equivalents” also encompassed in accordance with theinvention have an amino acid sequence which differs from SEQ ID NO:2 inat least one position, the modification in the sequence modifying themonooxygenase activity preferably only inconsiderably, i.e. by not morethan approximately ±90%, in particular ±50% or not more than ±30%. Thismodification can be determined using a reference substrate, such as, forexample, β-ionone, under standardized conditions (for example 0.1 to0.5M substrate, pH range 6 to 8, in particular 7; T=60 to 70° C., inparticular 65° C.).

“Functional equivalents” also encompassed in accordance with theinvention are homologs to the specifically disclosed proteins. They haveat least 60% homology, preferably at least 75% homology, in particularat least 85% homology, such as, for example, 90%, 95% or 99%, homologywith one of the specifically disclosed sequences, calculated by thealgorithm of Pearson and Lipman, Proc. Natl. Acad. Sci. (USA) 85(8),1988, 2444-2448.

Homologs of the proteins or polypeptides according to the invention canbe generated by mutagenesis, for example by point mutation or truncationof the proteins.

Homologs of the protein according to the invention can be identified byscreening combinatory libraries of mutants, such as, for example,truncated mutants. For example, a variegated library of protein variantscan be generated by combinatory mutagenesis at the nucleic acid level,such as, for example, by the enzymatic ligation of a mixture ofsynthetic oligonucleotides. There exists a multiplicity of processeswhich can be used for generating libraries of potential homologs from adegenerate oligonucleotide sequence. The chemical synthesis of adegenerate gene sequence can be carried out in a DNA synthesizer, andthe synthetic gene can be ligated into a suitable expression vector. Theuse of a degenerate set of genes makes it possible to provide, in amixture, all sequences which encode the desired set of potential proteinsequences. Methods for the synthesis of degenerate oligonucleotides areknown to the skilled worker (for example Narang, S. A. (1983)Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al., (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidsRes. 11:447).

“Functional equivalents” naturally also encompass P450 monooxygenaseswhich can be obtained from other organisms, for example from otherbacteria than those mentioned specifically herein, and naturallyoccurring variants. For example, areas of homologous sequence regionscan be identified by sequence alignment and equivalent enzymes can bedetermined with reference to the specific objects of the invention.

The substrates of group a) which can be oxidized in accordance with theinvention are optionally substituted heterocyclic mono-, bi- orpolynuclear aromatic compounds; in particular N-, O- or S-heterocyclicmono-, bi- or polynuclear aromatic compounds which can be oxidized orhydroxylated. They encompass for example two or three four- toseven-membered, in particular six- or five-membered, fused rings whereat least one, preferably all, of the rings have the aromatic characterand where at least one of the aromatic rings has one to three,preferably one, N-, O- or S-hetero atom attached to the ring. Ifappropriate, the entire ring structure may contain one or two furtheridentical or different hetero atoms. Furthermore, the aromatic compoundscan have 1 to 5 substituents attached to the ring carbon atoms or to thehetero atoms. Examples of suitable substituents are C₁- to C₄-alkyl suchas methyl, ethyl, n- or i-propyl or n-, i- or t-butyl or C₂- toC₄-alkenyl such as ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenylor 3-butenyl, hydroxyl and halogen such as F, Cl, and Br. Ifappropriate, the abovementioned alkyl or alkenyl substituents may alsohave a keto or aldehyde group; examples are propan-2-on-3-yl,butan-2-on-4-yl, 3-buten-2-on-4-yl. Nonlimiting examples of suitableheterocyclic substrates are, in particular, binuclear heterocycles suchas indole, N-methylindole and the analogs thereof which are substitutedon carbon atoms by one to three substituents, such as, for example,5-chloroindole or 5-bromoindole, and also quinoline and quinolinederivatives such as, for example, 8-methylquinoline, 6-methylquinolineand quinaldin; and benzothiophene and the analogs thereof which aresubstituted on carbon atoms by one to three substituents. Others whichmay be mentioned are trinuclear heteroaromatics such as acridine, andthe analogs thereof which are substituted on carbon atoms by one tothree substituents.

Substrates of group b) which can be oxidized in accordance with theinvention are optionally substituted mono- or polynuclear, in particularmono- or binuclear, aromatics such as benzene and naphthaline. Ifappropriate, the aromatic compounds can be mono- or polysubstituted andhave for example 1 to 5 substituents attached to the ring carbon atoms.Examples of suitable substituents are C₁- to C₄-alkyl such as methyl,ethyl, n- or i-propyl or n-, i- or t-butyl, or C₂- to C₄-alkenyl such asethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl or 3-butenyl,hydroxyl and halogen such as F, Cl, and Br. If appropriate, theabovementioned alkyl or alkenyl substituents may also have a keto oraldehyde group; examples are propan-2-on-3-yl, butan-2-on-4-yl,3-buten-2-on-4-yl. If appropriate, the aromatic ring can be fused to afour- to seven-membered nonaromatic ring. If appropriate, thenonaromatic ring can have one or two C-C double bonds, be mono- orpolysubstituted by abovementioned substituents and, if appropriate, haveattached to it one or two ring hetero atoms. Examples of particularlyuseful aromatics are mononuclear aromatics such as cumene, and binuclearsubstrates such as indene and naphthalene, and the analogs thereof whichare substituted on carbon atoms by one to three substituents.

Substrates of group c) which can be oxidized in accordance with theinvention are straight-chain or branched alkanes or alkenes having 4 to15, preferably 6 to 12, carbon atoms. Examples which may be mentionedare n-pentane, n-hexane, n-heptane, n-octane, n-nonane, n-decane,n-undecane and n-dodecane, and the analogs of these compounds which haveone or more branchings such as, for example, analogous compounds with 1to 3 methyl side groups; or the mono- or polyunsaturated, preferablymonounsaturated, analogs of the abovementioned alkanes.

Substrates of group d) which can be oxidized in accordance with theinvention are optionally substituted cycloalkanes and cycloalkenes.Examples are cyclopentane, cyclopentene, cyclohexane, cyclohexene,cycloheptane and cycloheptene. In this context, the ring structure canbe mono- or polysubstituted and can have attached to it for example 1 to5 substituents as defined above for compounds of groups a) and b). Anonlimiting example are ionones, such as α-, β- and γ-ionone, and thecorresponding methylionones and isomethylionones.

Substrates of group e) which can be oxidized in accordance with theinvention are straight-chain or branched, saturated or mono- orpolyunsaturated C₈-C₃₀-carboxylic acids, in particular monocarboxylicacids, or carboxylic acid derivatives thereof, such as esters andamides. Examples which can be mentioned are saturated monocarboxylicacids which can be hydroxylated terminally or subterminally (ω-1-, ω-2-or ω-3 position).

Subject matter of the invention are also nucleic acid sequences (single-and double-stranded DNA and RNA sequences) encoding one of the abovemonooxygenases, and their functional equivalents. Further nucleic acidsequences according to the invention are derived from SEQ ID NO:1 anddiffer therefrom by addition, substitution, insertion or deletion ofsingle or more than one nucleotides, with the continued encoding ofmonooxygenase with the desired spectrum of properties.

Also encompassed in accordance with the invention are those nucleic acidsequences which encompass what is known as silent mutations or which arealtered in accordance with the codon usage of a specific organism oforigin, or a host organism, in comparison with a specifically mentionedsequence, as are naturally occurring variants such as, for example,splice variants, thereof. Subject matter are also sequences which can beobtained by conservative nucleotide substitutions (i.e. the amino acidin question is replaced by an amino acid of the same charge, size,polarity and/or solubility).

The invention furthermore encompasses nucleic acid sequences whichhybridize with abovementioned coding sequences or are complementarythereto. These polynucleotides can be found by screening genomiclibraries or cDNA libraries and, if appropriate, amplified therefromusing suitable primers by means of PCR and subsequently isolated, forexample using suitable probes. Another possibility is the transformationof suitable microorganisms with polynucleotides or vectors according tothe invention, the multiplication of the microorganisms and thus theamplification of the polynucleotides, and their subsequent isolation.Moreover, polynucleotides according to the invention can also besynthesized chemically.

The property of being able to “hybridize” with polynucleotides isunderstood as the ability of a polynucleotide or oligonucleotide to bindto a virtually complementary sequence under stringent conditions, whileunspecific binding events between noncomplementary partners do not takeplace under these conditions. Here, the sequences should have 70-100%,preferably 90-100%, complementarity. The property of complementarysequences of being able specifically to bind to each other is exploitedfor example in the Northern or Southern blot technique, or for bindingprimers in PCR or RT-PCR. Oligonucleotides starting from a length of 30base pairs are normally employed for this purpose. Stringent conditionsare understood as meaning, for example for the Northern blot technique,the use of a wash solution at a temperature of 50-70° C., preferably60-65° C., for example 0.1×SSC buffer with 0.1% SDS (20×SSC: 3M NaCl,0.3M sodium citrate, pH 7.0) for eluting unspecifically hybridized cDNAprobes or oligonucleotides. As has been mentioned above, only nucleicacids with a high degree of complementarity remain bound to each otherin this process.

Subject matter of the invention are furthermore expression constructscontaining, under the genetic control of regulatory nucleic acidsequences, a nucleic acid sequence encoding a mutant according to theinvention; and vectors encompassing at least one of these expressionconstructs. Such constructs according to the invention preferablyencompass a promoter 5′-upstream and a terminator sequence 3′-downstreamof the coding sequence in question, and, if appropriate, furthercustomary regulatory elements, in each case linked operably to thecoding sequence. “Operable linkage” is understood as meaning thesequential arrangement of promoter, coding sequence, terminator and, ifappropriate, further regulatory elements in such a way that each of theregulatory elements can fulfill its function as intended upon theexpression of the coding sequence. Examples of sequences which can belinked operably are targeting sequences, and also translation and otherenhancers, polyadenylation signals and the like. Further regulatoryelements encompass selectable markers, amplification signals,replication origins and the like.

The natural regulatory sequence may still be present before the actualstructural gene, in addition to the artificial regulatory sequences. Ifappropriate, genetic modification can be used to switch off this naturalregulation and to increase or reduce the expression of the genes.However, the gene construct may also have a simpler structure, that isto say no additional regulatory signals are inserted before thestructural gene and the natural promoter together with its regulation isnot removed. Instead, the natural regulatory sequence is mutated in sucha way that regulation no longer takes place and gene expression isincreased or reduced. One or more copies of the nucleic acid sequencesmay be present in the gene construct.

Examples of useful promoters are: cos, tac, trp, tet, trp-tet, lpp, lac,lpp-lac, lacIq, T7, T5, T3, gal, trc, ara, SP6, l-PR or the l-PLpromotor, all of which are advantageously used in Gram-negativebacteria; and the Gram-positive promotors amy and SPO2, the yeastpromoters ADC1, MFa , AC, P-60, CYC1, GAPDH or the plant promotersCaMV/35S, SSU, OCS, lib4, usp, STLS1, B33, not, or the ubiquitin orphaseolin promotor. The use of inducible promoters, such as, forexample, light-inducible and, in particular, temperature-induciblepromoters, such as the P_(r)P_(l), promoter, is especially preferred.

In principle, all natural promoters together with their regulatorysequences can be used. In addition, synthetic promoters can also be usedadvantageously.

The abovementioned regulatory sequences are intended to make possiblethe targeted expression of the nucleic acid sequences and proteinexpression. Depending on the host organism, this may mean, for example,that the gene is expressed or overexpressed only after induction, orthat it is expressed and/or overexpressed immediately.

The regulatory sequences or factors can preferably have a positiveeffect on expression and thus increase or reduce it. Thus, theregulatory elements can be enhanced advantageously at thetranscriptional level by using strong transcription signals such aspromoters and/or enhancers. In addition, enhanced translation is alsopossible, for example by improving mRNA stability.

An expression cassette is prepared by fusing a suitable promoter to asuitable monooxygenase nucleotide sequence and to a terminator orpolyadenylation signal. Customary recombination and clone techniques areused for this purpose as they are described, for example, in T.Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)and in T. J. Silhavy, M. L. Berman and L. W. Enquist, Experiments withGene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1984) and in Ausubel, F. M. et al., Current Protocols in MolecularBiology, Greene Publishing Assoc. and Wiley Interscience (1987).

For expression in a suitable host organism, the recombinant nucleic acidconstruct or gene construct is advantageously inserted into ahost-specific vector which makes possible optimal expression of thegenes in the host. Vectors are well known to the skilled worker and canbe found, for example, in “Cloning Vectors” (Pouwels P. H. et al., Ed.,Elsevier, Amsterdam-New York-Oxford, 1985). In addition to plasmids,vectors are also understood as meaning all of the other vectors known tothe skilled worker, such as, for example, phages, viruses such as SV40,CMV, baculovirus and adenovirus, transposons, IS elements, phasmids,cosmids and linear or circular DNA. These vectors can be replicatedautonomously in the host organism or replicated chromosomally.

Recombinant microorganisms which, for example, are transformed with atleast one vector according to the invention and which can be employedfor producing the mutants can be generated with the aid of the vectorsaccording to the invention. Advantageously, the above-describedrecombinant constructs according to the invention are introduced into,and expressed in, a suitable host system. To do so, cloning andtransfection processes which the skilled worker is familiar with, suchas, for example, coprecipitation, protoplast fusion, electroporation,retroviral transfection and the like are preferably used in order toexpress the nucleic acids mentioned in the expression system inquestion. Suitable systems are described, for example, in CurrentProtocols in Molecular Biology, F. Ausubel et al., Ed., WileyInterscience, New York 1997.

Suitable as host organisms are, in principle, all the organisms whichmake possible an expression of the nucleic acids according to theinvention, their allelic variants, their functional equivalents orderivatives. Host organisms are understood as meaning, for example,bacteria, fungi, yeasts, plant or animal cells. Preferred organisms arebacteria such as those of the genera Escherichia, such as, for example,Escherichia coli, Streptomyces, Bacillus or Pseudomonas, eukaryoticmicroorganisms such as Saccharomyces cerevisiae, Aspergillus, highereukaryotic cells from animals or plants, for example Sf9 or CHO cells.

Successfully transformed organisms can be selected by means of markergenes which are also present in the vector or in the expressioncassette. Examples of such marker genes are genes for resistance toantibiotics and for enzymes which catalyze a coloring reaction whichcauses staining of the transformed cell. The latter can then be selectedby means of automatic cell sorting. Microorganisms which are transformedsuccessfully with a vector and which carry a relevant gene forresistance to antibiotics (for example G418 or hygromycin) can beselected by suitable liquid or solid media comprising antibiotics.Marker proteins which are presented on the cell surface can be utilizedfor selection by means of affinity chromatography.

The combination of the host organisms and the vectors which match theorganisms, such as plasmids, viruses or phages, such as, for example,plasmids with the RNA polymerase/promoter system, phages λ or μ or othertemperent phages or transposons and/or further advantageous regulatorysequences forms an expression system. For example, the term “expressionsystem” refers to the combination of mammalian cells such as CHO cells,and vectors such as pcDNA3neo vector, which are suitable for mammaliancells.

If desired, the gene product can also be expressed in transgenicorganisms such as transgenic animals, such as, in particular, mice orsheep, or transgenic plants.

Subject-matter of the invention are furthermore processes for therecombinant production of a monooxygenase according to the invention,wherein a monooxygenase-producing microorganism is grown, and theexpression of monooxygenase is, if appropriate, induced, and themonooxygenase is isolated from the culture. Thus, the monooxygenase canalso be produced on an industrial scale if so desired.

The recombinant microorganism can be grown and fermented by knownprocesses. For example, bacteria can be multiplied in TB or LB mediumand at a temperature of from 20 to 40° C. and a pH of from 6 to 9.Specific suitable culture conditions are described, for example, in T.Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989).

Unless the monooxygenase is secreted into the culture medium, the cellsare then disrupted and the enzyme is obtained from the lysate by knownprotein isolation processes. The cells can be disrupted by a process ofchoice selected from among high-frequency ultrasound, high pressure suchas, for example, in a French press, osmolysis, the action of detergents,lytic enzymes or organic solvents, homogenizers or a combination of morethan one of the processes stated.

Purification of the monooxygenase can be achieved by knownchromatographic processes such as chromatography with molecular sieve(gel filtration) such as Q-Sepharose chromatography, ion exchangechromatography and hydrophobic chromatography, and with other customaryprocesses such as ultrafiltration, crystallization, salting out,dialysis and native gel electrophoresis. Suitable processes aredescribed, for example, in Cooper, F. G., Biochemische Arbeitsprocessen[processes in Biochemistry], Verlag Walter de Gruyter, Berlin, New Yorkor in Scopes, R., Protein Purification, Springer Verlag, New York,Heidelberg, Berlin.

To isolate the recombinant protein, it is particularly advantageous touse vector systems or oligonucleotides which extend the cDNA by specificnucleotide sequences and thus encode modified polypeptide or fusionproteins in order to simplify purification. Such suitable modificationsare, for example, what is known as “tags” which have an anchoringfunction, such as, for example, the modification known as hexa-histidineanchor, or epitopes which can be recognized by antibodies as antigens(described, for example, in Harlow, E. and Lane, D., 1988, Antibodies: ALaboratory Manual. Cold Spring Harbor (N.Y.) Press). These anchors canserve for attaching the proteins to a solid support, such as, forexample, a polymer matrix, with which for example a chromatographiccolumn can be packed, or they can be used on a microtiter plate or anyother support.

These anchors can simultaneously also be used for recognizing theproteins. Others which can be used for recognizing the proteins arefurthermore customary labels such as fluorescent dyes, enzyme labelswhich, after reaction with a substrate, form a detectable reactionproduct, or radiolabels, alone or in combination with the anchors forderivatizing the proteins.

The invention furthermore relates to a process for the microbiologicaloxidation of organic compounds of the above type.

If the conversion is carried out with the recombinant microorganism, themicroorganisms are preferably first grown in the presence of oxygen andin a complex medium such as, for example, TB or LB medium, at a culturetemperature of approximately 20° C. or more and a pH of approximately 6to 9 until a sufficient cell density is reached. In order to bettergovern the oxidation reaction, the use of an inducible promoter ispreferred. After induction of the monooxygenase production, culturing iscontinued for 12 hours to 3 days in the presence of oxygen.

If, in contrast, the conversion in accordance with the invention iscarried out with purified or concentrated enzyme, the enzyme accordingto the invention is dissolved in a medium comprising an exogenoussubstrate (approx. 0.01 to 10 mM or 0.05 to 5 mM) and the conversion iscarried out at a temperature of approximately 10° C. or more and a pH ofapproximately 6 to 9 (such as, for example, adjusted with 100 to 200 mMphosphate buffer or Tris buffer), preferably in the presence of oxygen,and in the presence of a reducing agent, the substrate-comprising mediumadditionally comprising an approximately 10- to 100-fold molar excess ofreduction equivalents based on the substrate to be oxidized. Preferredreducing agent is NADPH.

In the substrate oxidation process according to the invention, oxygenwhich is present in the reaction medium or has been added is subjectedto enzymatic reductive cleavage. The reduction equivalents required areprovided by the reducing agent added (electron donor).

The oxidation product formed can then be separated from the medium andpurified in the customary fashion such as, for example, by extraction orchromatography.

The following nonlimiting examples describe specific embodiments of theinvention.

General Experimental Data

a) General Cloning Processes

The cloning steps carried out within the present invention such as, forexample, restriction cleavages, agarose gel electrophoresis,purification of DNA fragments, transfer of nucleic acids tonitrocellulose and nylon membranes, linking DNA fragments,transformation of E. coli cells, growing bacteria, phage multiplicationand sequence analysis of recombinant DNA were carried out as describedby Sambrook et al. (1989) loc. cit.

b) Polymerase Chain Reaction (PCR)

PCR was carried out by standard protocol with the following standardreaction mix:

8 μl of dNTP mix (200 μM), 10 μl of Taq polymerase buffer (10×) withoutMgCl₂, 8 μl MgCl₂ (25 μM), in each case 1 μl of primer (0.1 μM), 1 μl ofDNA to be amplified, 2.5 U of Taq polymerase (MBI Fermentas, Vilnius,Lithuania), demineralized water to 100 μl.

c) Culturing E. coli

Recombinant E. coli strains DH5α were cultured in LB-Amp medium(Tryptone 10.0 g, NaCl 5.0 g, yeast extract 5.0 g, Ampicillin 100 g/mlH₂O to 1000 ml) at 37° C. To this end, in each case one colony wastransferred from an agar plate to 5 ml LB-Amp by means of a loop. Afterculturing for approx. 18 hours at a shake frequency of 220 rpm, 400 mlof medium in a 2 1 flask were inoculated with 4 ml of culture. The P450expression in E. coli was induced after an OD578 value of between 0.8and 1.0 had been reached by heat-shock induction at 42° C. for three tofour hours.

d) Cell Disruption

Cell pellets with a biomass fresh weight of up to 15 g of E. coli DH5αwere defrosted on ice and suspended in 25 ml of potassium phosphatebuffer (50 mM, pH 7.5, 1 mM EDTA) or Tris/HCl buffer (50 mM, pH 7.5, 1mM EDTA). The E. coli cell suspension, which was cooled on ice, wasdisrupted by means of sonication for 3 minutes (Branson Sonifier W250,(Dietzenbach, Germany), power output 80 W, operating interval 20%).Prior to protein purification, the cell suspension was centrifuged for20 minutes at 32.500 g and filtered through a 0.22 mm Sterivex-GP filter(Millipore), yielding a crude extract.

EXAMPLE 1

Cloning and Expression of P450 from Thermus thermophilus HB27 and itshis-tag Derivatives

1. Cloning of P450 from Thermus thermophilus HB27

The coding P450 sequence (blunt ended) was cloned into the HincIIcleavage site of plasmid pTZ19R (MBI Fermentas). The coding P450sequence was amplified from the resulting plasmid TTHB66 with the aid ofPCR. The following primers were used for this purpose:

-   a) 30-mer sense oligonucleotide comprising the NdeI cleavage site    (italicized) as part of the P450 ATG start codon:    -   5′-CGAAGCTCATATGAAGCGCCTTTCCCTGAG (SEQ ID NO:7).-   b) 30-mer antisense oligonucleotide comprising the EcoRI cleavage    site (italicized) as part of the TGA stop codon:    -   5′-GCGAATTCACGCCCGCACCTCCTCCCTAGG (SEQ ID NO:8).

The resulting fragment was cloned into the NdeI cleavage sites of vectorpCYTEXP1 (plasmid with the temperature-inducible P_(R)P_(L) promotersystem of bacteriophage λ (Belev T. N., et al., Plasmid (1991) 26:147))and transformed into E. coli DH-5α (Clontech, Heidelberg).

E. coli DH-5α, comprising the plasmid of interest, was inoculated intoLB medium in the presence of Ampicillin and the culture wasincubated-overnight at 37° C. Some of the sample was inoculated intofresh LB medium (in the presence of Ampicillin), and the resultingculture was grown at 37° C. to OD=0.9. Induction was affected by raisingthe temperature to 42° C. over a period of 24 hours. The change in theP450 content during expression was determined by measuring the COdifference spectrum.

Expression time [h] ΔA₄₅₀₋₄₉₀ P450 concentration [μM] 4 0.092 0.056 80.176 0.106 24 0.106 0.0642. Cloning P450 from Thermus thermophilus HB27 with N-terminal his tag

The coding P450 sequence was amplified from plasmid TTHB66 by PCR usingthe following primers:

-   (a) 50-mer sense oligonucleotide comprising the NdeI cleavage site    (italicized) as part of the P450 ATG start codon and the tag-coding    codons (underlined):    -   5′-CGAAGCTCATATGCATCACCATCATCATCACAAGCGCCTTTC (SEQ ID NO:9);-   (b) 30-mer antisense oligonucleotide comprising the EcoRI cleavage    site (italicized) as part of the TGA stop codon:    -   5′-GCGAATTCACGCCCGCACCTCCTCCCTAGG (SEQ ID NO:8).

The resulting fragment was cloned into the NdeI and EcoRI cleavage sitesof vector p-CYTEXP1 and expressed in E. coli DH-5α.

E. coli DH-5α, comprising the plasmid of interest, was inoculated intoLB medium in the presence of Ampicillin and the culture was incubatedovernight at 37° C. Some of the sample was inoculated into fresh LBmedium (in the presence of Ampicillin), and the resulting culture wasgrown at 37° C. to OD=0.9. Induction was affected by raising thetemperature to 42° C. over a period of 24 hours. The change in the P450content during expression was determined by measuring the CO differencespectrum.

Expression time [h] ΔA₄₅₀₋₄₉₀ P450 concentration [μM] 4 ND ND 8 0.0970.073 24 0.111 0.0733. Cloning P450 from Thermus thermophilus HB27 with C-terminal his tag

The coding P450 sequence was amplified from plasmid TTHB66 by PCR usingthe following primers:

-   (a) 30-mer sense oligonucleotide comprising the NdeI cleavage site    (italicized) as part of the P450 ATG start codon:    -   5′-CGAAGCTCATATGAAGCGCCTTTCCCTGAG (SEQ ID NO:7)-   (b) 47-mer antisense oligonucleotide comprising the EcoRI cleavage    site (italicized) as part of the TGA stop codon and the underlined    tag-encoding part sequence:    -   5′-CGGAATTCAGTGATGATGATGGTGATGCGCCCGCACCTCCTC (SEQ ID NO:10).

The resulting fragment was cloned into the NdeI and EcoRI cleavage sitesof vector p-CYTEXPl and expressed in E. coli DH-5α.

E. coli DH-5α, comprising the plasmid of interest, was inoculated intoLB medium in the presence of Ampicillin and the culture was incubatedovernight at 37° C. Some of the sample was inoculated into fresh LBmedium (in the presence of Ampicillin), and the resulting culture wasgrown at 37° C. to OD=0.9. Induction was affected by raising thetemperature to 42° C. over a period of 24 hours. The change in the P450content during expression was determined by measuring the CO differencespectrum.

Expression time P450 concentration [h] ΔA₄₅₀₋₄₉₀ [μM] 4 ND ND 8 0.10.075 24 ND ND

EXAMPLE 2

Determination of the Thermostability of Thermus thermophilus P450 inComparison with P450 BM3

The two enzymes were incubated in each case for 30 minutes in Tris/HClbuffer pH 7.5, 25 mM, at different temperatures. The reaction mixtureswere subsequently cooled and the P450 concentration was determinedspectrometrically. The results are compiled in the table which followsand shown in FIG. 2 in the form of a graph.

Temperature [° C.] 30 40 50 60 P450 concentration P450 thermus 100 89 2922 [%] P450 BM3 92 63 0 0

As can be seen from the test results, the enzyme according to theinvention has a significantly higher thermostability after incubationfor 30 minutes at all temperatures.

EXAMPLE 3

Biotransformation Experiments

It has hitherto not been possible unambiguously to identify theendogenous redox partner for the T. thermophilus cytochrome P450[lacuna] according to the invention. However, enzyme activity wasobserved, for example, during the hydroxylation of β- and/or α-ionone.With β-ionone as substrate, conversion into a main product was observed,whereupon α-ionone was converted into a product mixture. A comparisonwith synthetic standards revealed that the main product of theconversion of β-ionone is 4-hydroxy-β-ionone.

Precultures of T. thermophilus [5 ml of Tt medium (2 g of yeast extract,1 g of tryptone, 1 g of NaCl in 500 ml of deionized water)] wereinoculated from agar plate cultures and incubated for 24 hours at 65° C.with shaking (150 rpm). Subsequently 100 ml of the Tt medium wereinoculated with the preculture and incubated at 65° C. with shaking.

β-Ionone (107 μl/ml of culture) was added to each culture after 24hours. Cultivation was continued for 78 hours. The cells were removed bycentrifugation and the supernatant was extracted with diethyl ether. Theextract was analyzed by GC and TLC. Control cultures without substratewere prepared and analyzed under identical conditions.

1. An isolated cytochrome P450 monooxygenase having the amino acidsequence of SEQ ID NO: 2 or; an amino acid sequence having at least 95%homology with SEQ ID NO:
 2. 2. The isolated cytochrome P450monooxygenase as claimed in claim 1 which is from bacteria of the genusThermus sp.
 3. The isolated cytochrome P450 monooxygenase as claimed inclaim 2 which is from a bacterium of the species Thermus thermophilus.4. A process for the microbiological oxidation of an organic compound,wherein this compound is converted with at least one isolated cytochromeP450 monooxygenase as claimed in claim 1 and wherein the compound isselected from among: optionally substituted N-, O- or S-heterocyclicmono-, bi- or polynuclear aromatic compounds; optionally substitutedmono- or polynuclear aromatics; straight-chain or branched alkanes andalkenes; optionally substituted cycloalkanes and cycloalkenes; andaliphatic (terminally saturated) carboxylic acids.
 5. A bioreactorencompassing the isolated cytochrome P450 monooxygenase as claimed inclaim 1.